**3.1 Effect of % deacetylation (DD) of chitosan**

328 Biogas

Chitosan has been largely employed in many areas, such as photography, biotechnology, cosmetics, food processing, biomedical products (artificial skin, wound dressing, contact lens, etc.) and in a system for controlled liberation of medicines (capsules and microcapsules). In addition, chitosan has been used as a flocculant for the removal of metallic and colouring ions from industrial effluents by bonding the micro-floc particles together to form larger, denser

Chitosan is a natural polysaccharide whose structure is similar to extracellular polymeric substances (ECP). ECP are widely known to assist anaerobic cell aggregation. Polymeric chains of ECP enhance flocculation by bridging microbial cells to form an initial microbial nucleus which is the first step in microbial granulation. There are many hypotheses to explain adhesion and aggregation processes by ECP. For example, in one hypothesis, ECP production is thought to occur prior to adhesion and the appearance of polymer materials at the initial site of contact between microbial cells is believed to be caused by the migration of polymer molecules onto the cell surface. In another hypothesis, ECP production is thought to occur after adhesion. In this case, it is believed that bacterial adhesion provides a favorable physiological condition for ECP excretion (El-Mamouni et al., 1998; Liu et al., 2002;

Chitosan is obtained by partial deacetylation of chitin (de Alvarenga et al., 2010). Chitin is a -(1→4)-linked polymer of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine) which exists in the exoskeletons of insects, crustaceans and the cell walls of fungi and algae. Basically, deacetylation involves the replacement of acetyl groups in the molecular chain of chitin by complete amino groups (NH2). Chitosan is a mixture of straight-chain copolymers of N-acetyl-D-glucosamine and D-glucosamine of varying degrees of deacetylation (DD), i.e., with varying average numbers of D-glucosamine units per 100 monomers (Khan et al., 2002; Sabnis & Block, 1997). Chitosan also has the advantage that it is naturally

Chitosan is insoluble in water, organic solvents and aqueous bases, but it is soluble after stirring in acids such as acetic, nitric, hydrochloric, perchloric and phosphoric acids (de Alvarenga et al., 2010). The glucosamine moieties in chitosan carry free amine groups that are protonated in an acidic environment. The amount and the positions of the glucosamine determine the charge and the charge distribution in the chitosan molecule. Changes in charge density have an effect on the dissolution and binding properties of chitosan (Domard, 1996). The degree of deacetylation also controls the degree of crystallinity and hydrophobicity of chitosan (Vander Lubben et al., 2003). Chitosan enhances the flocculation of sludge, and the flocculation efficiency depends on both DD and molecular weight (MW).

biodegradable and therefore should have little adverse affect on human health.

NaOH Deacetylation

Fig. 1. Deacetylation of chitin to chitosan

flakes that are easier to separate (de Alvarenga et al., 2010; Renault et al., 2009).

**2. Chitosan as flocculants** 

Show et al., 2006a).

pH 7 is a typical starting pH in a UASB and most other anaerobic digesters (Lettinga et al., 1980). Kaseamchochoung et al. (2006) investigated the effect of %DD of chitosan on anerobic flocculation by using chitosan with different degrees of deactylation: M85 (DD = 85%) and M70 (DD = 70%) at pH 7. Their experimental procedure was as follows. In the flocculation assay, an initial sludge suspension was transferred into a beaker and a chitosan stock solution was added to achieve a concentration of 0 to 45 mg chitosan/g oven-dried (o.d.) sludge. The suspension was then stirred. The pH of the suspension was adjusted to 5, 6, or 7, with either 1% acetic acid or 3% sodium carbonate, depending on the pH of chitosan added to the suspension. After continuous mixing, the turbidity of supernatant was determined using a turbidimeter. The flocculation was calculated from the decrease in turbidity of supernatant after the treatment with chitosan compared with a reference without chitosan.

Fig. 2. Flocculation and zeta potential as a function of chitosan concentration in sludge suspension at pH 7 with ionic strength of 0.1 M (from Kasemchochoung et al., 2006. Reprinted with permission from *Water Environment Research*. Volume 78, No. 11, pp. 2211 to 2214, Copyright © 2006 Water Environment Federation, Alexandria, Virginia.)

Enhancing Biogas Production and UASB Start-Up by Chitosan Addition 331

Similar results were obtained by Roussy et al. (2004). They studied chitosan efficiency at three different pH values (pH 5, 6.3, and 9). They found that a lower chitosan dosage (87% DD) was required at pH 5, while a significantly higher dosage of chitosan was required at pH 9 to obtain a residual turbidity below a fixed limit of 5 formalin turbidity units. Their explanation was that two possible mechanisms were possible at pH 5—(a) coagulation by charge neutralization and (b) flocculation by entrapment in the polymer network. However, at pH 9 only the latter mechanism is possible, but its effect can only be significant at a high

Kaseamchochoung et al. (2006) found that both chitosan M70 and M85 were able to flocculate anaerobic sludge even when the system pH dropped to 5. A small degree of restabilization was observed after the charge neutralization point (CPN). That is, the percentage of flocculation dropped only slightly after the CPN, whereas zeta potential values became positive. A possible explanation given in Kaseamchochoung et al. (2006) is that the charge density of chitosan is greatly influenced by pH (Strand et al., 2001). Because the intrinsic pKa of chitosan is close to 6.5, most amine groups are protonated at pH 5, but become significantly less protonated when the pH increases. The polymer is therefore more highly positively charged at pH 5 than at pH 7. At pH 7, chitosan with 70%DD contains a lower charge density than chitosan with 85%DD, and the performance of chitosan (70%DD) would be noticeably lower at a low chitosan dosage (Fig. 2). Kaseamchochoung et al. (2006) suggested that charge density may play an important role in the flocculation mechanism and that this is not surprising because electrostatic forces are typically the main cause of polyelectrolyte adsorption on an oppositely charged surface. They concluded that chitosan has the potential to be used as an effective cationic bioflocculant, which is able to function either in acidic or neutral conditions, and that only relatively small amounts of chitosan (less

Fig. 4. Percent flocculation as a function of chitosan M70 concentration in sludge suspension at different pH values and ionic strengths (from Kasemchochoung et al., 2006. Reprinted with permission from *Water Environment Research*. Volume 78, No. 11, pp. 2211 to 2214,

Copyright © 2006 Water Environment Federation, Alexandria, Virginia.)

chitosan concentration.

than 4 mg/g dried sludge) are required.

Kaseamchochoung et al. (2006) found that at a low concentration (2 mg chitosan/g o.d. sludge) chitosan M85 gave approximately 90% flocculation, whereas M70 gave only approximately 80% flocculation (Fig. 2). However, at a concentration of 4 mg chitosan/g o.d. sludge the flocculation efficiencies of M70 and M85 became approximately equal at 95% flocculation and then remained approximately equal up to concentrations of 45 mg chitosan/g o.d. sludge (Fig. 2).
